CD2AP突变对肾小球足细胞骨架蛋白F-actin分布的影响

Effects of CD2AP mutation on distribution of cytoskeletal protein F-actin in podocytes

目的构建突变型CD2相关蛋白(CD2AP)荧光表达载体并转染肾小球足细胞,观察细胞内骨架蛋白F-actin分布的变化。方法定点诱变真核表达质粒pcDNA6-V5-CD2AP致CD2AP第2外显子160G>A杂合突变,测序鉴定。利用野生型和突变型质粒和pEGFP-C1荧光表达载体,分别构建野生型和突变型重组质粒pEGFP-C1-CD2AP荧光表达载体并转染小鼠肾小球足细胞,荧光显微镜观察处于不同细胞周期的足细胞内F-actin分布情况。结果测序结果显示,pcDNA6-V5-CD2AP真核表达载体CD2AP的2号外显子第160位碱基G诱变为A,而其他碱基未发生改变。在细胞分裂间期,未转染足细胞内F-actin平行排列成纤维束;野生型重组质粒转染足细胞内F-actin在细胞核周围呈点、块状浓聚;而突变型重组质粒转染足细胞内F-actin主要在细胞膜上勾出轮廓。在细胞分裂中期,野生型重组质粒转染足细胞内F-acin纤维丝呈粗纤维状,环细胞浓聚;突变型重组质粒转染足细胞则仅见胞质内F-acin纤维丝增多,部分区域浓聚。结论成功构建野生型和突变型重组CD2AP荧光表达载体。CD2AP基因160G>A杂合突变使肾小球足细胞分裂间期和分裂中期的F-actin纤维骨架发生改变。

Objective To construct mutant CD2-associated protein （CD2AP） fluorescence expression vector and transfect into podocytes, and investigate the distribution of F-actin in podocytes. Methods Eukaryotic expression plasmid pcDNA6-V5-CD2AP site-directed mutagenesis for 160G〉A heterozygous mutation in exon 2 of CD2AP was conducted, and identification was performed by sequencing. Wild-type and mutant recombinant plasmid pEGFP-C1-CD2AP fluorescence expression vectors were constructed with wild-type and mutant plasmid and pEGFP-C1 fluorescence expression vectors, respectively, and mouse podocytes were transfected. The distributions of F-actin in podocytes at different cell cycles were observed by fluorescence microscopy. Results Sequencing analysis revealed that sitedirect mutagenesis of 160 G in exon 2 of pcDNA6-V5-CD2AP eukaryotic expression vector CD2AP to A was performed, while there was no changes in the other bases. In the interphase of cell division, F-actin in untransfected podocytes lined in parallel as fiber bundle. F-actin in podocytes transfected with wild-type recombinant plasmid displayed thick, short dot-like structures mainly around the nuclei, while F-actin in podocytes transfected with mutant recombinant plasmid expressed along the cytoplasm membrane. In the metaphase of cell division, F-actin filaments transfected with wild-type recombinant plasmid aggregated encircling podocytes, while those transfected with mutant recombinant plasmid only emerged as a number increase in cytoplasm with some regional aggregation. Conclusion Wild-type and mutant recombinant CD2AP fluorescence expression vectors are successfully constructed. CD2AP gene 160G〉A heterozygous mutation may lead to the cytoskeletal changes of F-actin in the interphase and metaphase of cell division of podocytes.